A Technique for the Prolonged Incubation of Neuronal Tissue of the Retina
Transcrição
For retinal whole mount preparation, immediately enucleate the eyes from a euthanized animal. Make a small cut along the ora serrata, and place the eyes in either Ames Media or aCSF with carbogen supply at room temperature. After that, immediately remove the cornea, lens, and vitreous by cutting along the ora serrata with small scissors, and remove them with forceps. Place the tissue in the incubation system at room temperature.
To remove the inner limiting membrane, transfer the eye cup containing the retina to a small glass jar containing papain, L-cysteine, EDTA, and DNAse at 37 degrees Celsius for 20 minutes. Apply carbogen to the solution through the lid, but do not bubble. Stop the enzymatic digestion by placing the tissue in an ovomucoid and BSA solution for 10 minutes in Earl's BSS. Then, wash the tissue thoroughly with aCSF.
Subsequently, transfer the tissue to the incubation system and reduce the temperature to about 15 to 16 degrees Celsius. Following that, transfer the retinal tissue to the microscope; isolate the retina from the eye cup, and cut it into four pieces with a razor blade. If the entire retina is required, make four small cuts in the periphery of the retina to allow it to lie flat.
This tissue can now be transferred to the microscope for electrophysiology, or maintained in the Braincubator. The tissue is maintained in the main chamber containing the probes for pH and temperature measurements. A second chamber is isolated from the main chamber and is exposed to 1.1 watts UVC light in order to irradiate bacteria floating in the solution. Use a peristaltic pump to circulate aCSF through the two chambers, and a Peltier thermoelectric cold plate to either cool or heat the main chamber.
